1. Field of the Invention
The invention relates to a culture technology of a cell or tissue employed by a tissue engineering that is applied to a cell or tissue engineering or genetic treatment, particularly to a method of and an apparatus for cultivating a cell or tissue (hereinafter referred to as culture method and culture apparatus) for use in an in vitro culture of a cell or tissue that is needed for restoring a damaged tissue of human body.
2. Description of the Related Art
There are following methods for restoring a damaged tissue or a pathogenic part of a living body. The first method is to substitute the damaged tissue or pathogenic part for materials other than a living body such as plastic, metal, ceramic as restoring means of the damaged tissue or the pathogenic part. As substitutable materials, there are ceramics and stainless steel for bones, a polyethylene resin for joints, and a vinyl resin for blood vessels. Second method is to substitute the damaged tissue or pathogenic part for parts of other animals or the different position of the living body. As the substitutable tissue in the second method, there are, for example, skins. The third method is to transplant of internal organs of other people.
In the first method, there is a drawback that the materials other than the living body such as plastic, metal, ceramic need to be substituted periodically by others when they are worn or consumed or materials separated from the materials other than the living body by the wear thereof affects adversely on the living body. Further, in a blood vessel made of a synthetic polymer, there is a report that an interior of the blood vessel is clogged when it is used for a long period of time. In the third method, if there is no donor for supplies his or her internal organs to be transplanted, it is impossible to carry out the third method. Even if the third method is carried out, there still remains a problem of immunological rejection between internal organs of two people.
Accordingly, a method of restoring a damaged tissue or a pathogenic part of a living body that is expected to be carried out is to substitute the damaged part of a cell or tissue by a cell or tissue that is obtained by cultivating a cell or tissue in vivo or in vitro. It is reported in current researches that there is a possibility in many tissues such as skins, cartilage, bones, blood vessels, livers, and pancreas. If a cell or tissue derived from a living body is cultivated inside or outside the living body of a patient, and the cell or tissue obtained by the culture is applied to the restoration of a damaged part, a tissue can be regenerated in the body, and further the tissue applied to the restoration would have gene of the patient per se, there does not occur immunological rejection, and further, a chemical substance such as synthetic polymer other than a living material does not adversely affect a living body, thereby realizing an ideal treatment.
There has been proposed and disclosed as a technology of this type in Japanese Patent Laid-Open Publication No. 9-313166 entitled xe2x80x9cDEVICE FOR CULTURING CELLxe2x80x9d. This technology needs to disassemble into each part every culture, to clean, to sterilize, and then reassemble the apparatus, resulting in a risk of contamination by bacteria after sterilization. Although each part of the apparatus can be assembled for preventing contamination by bacteria so as to perform a sterilization treatment by an autoclave (absolute pressure 2 atm. at 121xc2x0 C.), this technology can not be employed in view of the contamination by bacteria because a pump and a pressure sensor include many electronic devices, a specific resin and oil. Accordingly, parts of the pump and pressure sensor are disassembled while only a passage through which a culture medium passes is taken out and is sterilized by chemicals, and other parts are sterilized by the autoclave, thereafter the pump and pressure sensor are assembled together with the apparatus, resulting in much labor and the increase of risk of contamination by various bacteria. Further, in the culture using an incubator (culture vessel), a pump or a controller is subjected to an adverse affection by a temperature or humidity, and also all the devices can not be accommodated in the incubator having a limited capacity. Accordingly, it is necessary to assemble the culture apparatus in a state where the incubator communicates with an open air for allowing piping, a power supply and a controlling electric wire to pass through a through hole of the incubator. Still further, since a pressure is applied to an entire circuit of a culture medium, the entire culture apparatus including parts of the pump and piping shall have a pressure resistant construction. As a result, it is very difficult to place the apparatus at high pressure e.g. not less than 1 MPa, and even if a high pressure is applied to the apparatus, the apparatus shall be high pressure resistant as a whole, resulting in a problem of high cost.
More still further, there is a research reported by Dr. Shuichi MIZUNO et al. in Harvard Medical School that a tissue of a living body is cultivated by applying a pressure to the living body as physical stimulation (see Materials Science and Engineering C6 (1998) 301-306). According to this research, a culture apparatus is formed as illustrated in FIG. 26. Each constituent and function thereof in this culture apparatus is described now.
A pump 400 has a role to circulate a culture medium 402 therein and to pressurize the interior of a culture chamber 404 to supply a hydraulic pressure to a cell 406 or tissue, and it is formed of a pump for use in a liquid chromatograph, and further it has a control unit built therein for flowing a given amount of fluid.
A back pressure regulator 408 allows a pressure to escape through a valve 410 by opening the valve 410 when a pressure exceeds a pressure to be applied to the cell 406 or tissue exceeds so as to hold the pressure inside the culture chamber 404 constant. The back pressure regulator 408 is selectively provided in a circuit 426, described later, depending on a pressure to be applied to the cell 406.
The culture chamber 404 forms a space for cultivating the cell 406 or tissue, and a scaffold 412 formed of a sponge made of a collagen in which the cell 406 or tissue is transplanted is accommodated in the space. The cell 406 or tissue grows on the scaffold 412 formed of a sponge made of a collagen. A pressure sensor 414 detects a pressure inside the culture chamber 404 while a pressure monitor 416 indicates the pressure detected by the pressure sensor 414. The pump 400 is controlled by the pressure detected by the pressure sensor 414 and it stops its operation when the detected pressure increases to a large extent.
A culture medium tank 418 stores therein the culture medium 402 adapted for the cell 406 or tissue to be cultivated and the culture medium 402 comprises e.g., amino acids, saccharides, salts, and so forth. The culture medium tank 418 communicates with an open air through a vent tube 422 that penetrates a closed stopper 420, and a vent filter 424 prevents the culture medium 402 from being contaminated by an open air.
The culture apparatus is accommodated in an incubator forming a hermetically sealed space. The incubator is a space for forming a pleasant cultivating atmosphere and it is maintained under the optimum temperature, humidity and gas concentration (oxygen and carbon dioxide) that is optimized for the cell or tissue. The culture medium 402 is filled in the circuit 426 by the pump 400 and circulated therein. The oxygen and carbon dioxide are soluble in the culture medium 402 after they pass through the vent filter 424, and the culture medium 402 is kept under appropriate oxygen concentration and carbon dioxide concentration. When the pump 400 is operated, a pressure inside the culture chamber 404 gradually increases. When the pressure exceeds a given value set by the back pressure regulator 408, the valve 410 of the back pressure regulator 408 is opened to discharge the culture medium 402 so that a pressure of the culture medium 402 is decreased by the amount of the discharged culture medium 402, thereby shutting the valve 410. With the repetition of these operations, a fixed pressure is maintained, and at the same time a fixed amount of the culture medium 402 is repetitively circulated. The cell 406 or tissue grows while it is subjected to such pressure application stimulation.
Although a fixed pressure is maintained in this culture apparatus, the increase and decrease of a pressure can not be repeated. Since the increase of the pressure is made by the pump 400, the rate of increase of the pressure is determined by the capacity of the pump 400. If the amount of circulation of the culture medium 402 increases, the rate of increase of the pressure becomes fast, while if the amount of circulation of the culture medium 402 decreases, the rate of increase of the pressure becomes slow. Accordingly, if a pressure cycle is continuously repeated, there is a method of providing a bypass 432 having a bypass valve 428 and an orifice valve (needle valve) 430 in parallel with a back pressure regulator 408 as shown in FIG. 27 to decrease the pressure. In this method, although the pressure can be decreased, there is a drawback that it takes a long time for one cycle, and the setting of a repetitive cycle and the circulation amount of the culture medium 402 can not be independent from each other, and further the regulation of the orifice valve 430 is finely controlled so as to render the rate of decrease of the pressure unstable.
Since the culture apparatus has to be assembled after each component thereof is disassembled, cleaned and sterilized every performance of culture, there is the risk of contamination by the bacteria after sterilization. Although it is possible to subject the assembled culture apparatus to a sterilization treatment by an autoclave (absolute pressure 2 atm. at 121xc2x0 C.), the pump and pressure sensor can not be sterilized because they include many electronic devices, specific resin and oil. Accordingly, under the existing circumstances, only a passage through which the culture medium 402 passes is taken out while each part of the pump and pressure sensor is disassembled, and each part is subjected to a sterilization by chemicals. The other parts of the pump and pressure sensor are subjected to a sterilization by an autoclave, then the pump, pressure sensor and the culture apparatus are respectively assembled, resulting in much labor and a risk of contamination by various bacteria.
Although oxygen and carbon dioxide are taken in the culture medium 402 through a filter, they are directly taken in from an ambient atmosphere, resulting in a risk of the contamination by bacteria. Further, although the culture apparatus is accommodated in an incubator, a pump unit and a pressure monitor is susceptible to a temperature and humidity so that the pump unit and pressure monitor are hardly accommodated in the incubator in view of their capacities. Accordingly, it is necessary to assemble the culture apparatus by allowing a tube for piping, a power supply and an electric wire for controlling to pass through a through hole of the incubator so that the inside and outside thereof are connected to each other.
Since a pressure is set by selecting a back pressure regulator depending on a set pressure, when the setting of a pressure is changed, the back pressure regulator is replaced by another one, resulting in much labor and a risk of contamination by various bacteria.
When a pressure cycle is changed, a pressure at a low pressure side can not be set in the culture apparatus in FIG. 27, but the pressure can be regulated by the orifice valve 430 so that the set pressure is varied by the quantity of circulated flow through the pump 400 even if a pressure can be regulated to some extent by the orifice valve 430.
As set forth above, in the conventional method of cultivating a cell or tissue of a living body, the cell is cultivated under a condition where a temperature, a humidity, a concentration of carbon dioxide and a concentration of oxygen are optimally set in an incubator. In such a culture in the incubator, it is a two-dimensional culture on a laboratory dish, and a three-dimensional culture has been now tried. Further, in such a culture method, a culture medium, cell or tissue that is exposed by an open air is prone to contamination by bacteria, so that stable culture is hardly performed.
Further, since the cell of a living body is always placed under physical stimulation and the stimulation indirectly affects the control of metabolism of a cell, a cell division cycle, a concentration gradient and dispersion of living body stimulation, and hence stable culture is hardly realized, and further, it has been hard to set or change the amount, variation, cycle of the physical stimulation. More still further, delicate set and regulation of a pressure are needed in culture, which requires a skill of a person in charge of culture.
Accordingly, in the conventional in vitro culture of a living cell, it takes time for a cell to grow to the same size as a cell to be restored, and hence there occurs a case where a normal culture is marred by contamination.
It is therefore an object of the invention to provide a method of and an apparatus for cultivating a cell or tissue capable of preventing the cell or tissue from being contaminated, and of realizing an efficient in vitro culture.
According to the invention, a culture position (culture chamber) is installed under an arbitrarily controlled environment such as an environment mimicking the living body, a culture medium is supplied while a cell or tissue is held in the foregoing culture position so that the cell or tissue is cultivated at the culture position under an ideal environment, whereby the cell or tissue is prevented from contamination and an efficient in vitro culture thereof can be realized.
To achieve the above object, the method of cultivating a cell or tissue according to the first aspect of the invention is characterized in comprising holding a cell or tissue of a living body at a specific culture position, setting the cell or tissue under an environment mimicking the living body, supplying a culture medium to the cell or tissue, and cultivating the cell or tissue at the culture position.
That is, it is ideal that a tissue necessary for restoring a damaged tissue of a living body and so forth is used by a cell or tissue of the same living body. It is necessary to cultivate to perform an in vitro culture using a cell or tissue collected from the living body to realize it. It is important for the in vitro culture that the contamination is prevented and a culture environment likewise a living body, namely, an environment mimicking the living body is artificially realized. For this end, a culture position is set under an environment formed artificially and the cell or tissue is held at a culture position and the culture medium is supplied to realize the in vitro culture of the cell or tissue. An environment means living condition including internal or external stimulation needed for maintaining a life healthy on the basis of a living body formed of a cell or tissue. The culture medium includes a nutrition source needed for maintaining the life of a cell or tissue and growing it. In this case, the supply of the culture medium applies a hydraulic pressure and physical stimulation to the cell or tissue, so that the cell or tissue is susceptible to metabolism functions, cell division cycle, concentration gradient or dispersion of living body stimulation so that the culture is enhanced. As a result, it is possible to cultivate the cell or tissue which is close to a tissue in a living body and easily fusible with a tissue in a living body.
The method of cultivating a cell or tissue according to the second aspect of the invention is characterized in comprising holding a cell or tissue of a living body at a specific culture position (culture chamber), setting the cell or tissue under an environment mimicking the living body, supplying continuously or intermittently a culture medium to the cell or tissue by way of a culture circuit (culture circuit), applying a pressure which is varied continuously, a pressure which is varied intermittently or a pressure which is varied periodically to the cell or tissue, and cultivating the cell or tissue at the culture position.
The setting of the culture position and environment are the same as mentioned above. The culture medium is continuously or intermittently supplied to the cell or tissue that is set at the culture position through the culture circuit. When the culture medium is supplied to the cell or tissue through the culture circuit that is separated or intercepted from the outside, the mode of supply of the culture medium can be made continuous or intermittent, and also the prevention of contamination can be achieved. It is possible to mimic a living body and to cultivate the cell or tissue efficiently by controlling the mode of the supply of the culture medium, corresponding to a living body environment. A desired pressure acts on the cell or tissue under culture thereof to apply physical stimulation. The mode of application of pressure is varied continuously, intermittently or periodically to mimic a living body, and apply a physical or mechanical strength needed for a living body such as flexibility or durability needed for the cell or tissue to be cultivated. Accordingly, it is possible to contribute to the culture of a cell or tissue which is ideal or practical, corresponding to a living body at a specific position of a living body, namely, contribute to cultivate the cell or tissue which is close to a tissue in a living body and easily fusible with a tissue in a living body.
The method of cultivating a cell or tissue according to the third aspect of the invention is characterized in further providing holding means for holding the cell or tissue to be cultivated at the culture position in a suspending or non-suspending state in the culture medium. That is, an experiment confirmed that the cell or tissue to be cultivated is held in a static state, that is needed for enhancing a culture efficiency.
The method of cultivating a cell or tissue according to the fourth aspect of the invention is characterized in that the holding means employs a hydro-gel for holding the cell or tissue to be cultivated at the culture position in a suspending state in the culture medium or a scaffold for holding the cell or tissue and absorbing the cell or tissue when it grows. That is, the cell or tissue to be cultivated may be held in any way, and hydro-gel or scaffold is one example of holding the cell or tissue in this case. The hydro-gel is means for wrapping and holding the cell or tissue to be cultivated in a suspending state, and the cell or tissue can be taken out from the hydro-gel when the culture is completed. Further, the scaffold may comprise a porous body formed of protein, and the cell or tissue to be cultivated is held by the scaffold and absorbs the scaffold as a nutrition as it grows.
The method of cultivating a cell or tissue according to the fifth aspect of the invention is characterized in that the culture medium includes one or not less than two of amino acids of various types, saccharides, salts and protein. That is, it is possible to use the culture medium corresponding to the cell or tissue to be cultivated, for example, it is possible to use one of amino acids of various types, saccharides, salts and protein or not less than two of materials selected therefrom or all of these materials. The selection of the culture medium is an essential element for efficient culture or for forming the cell or tissue with high quality.
The method of cultivating a cell or tissue according to the sixth aspect of the invention is characterized in that the environment mimicking the living body under which the cell or tissue is cultivated is set depending on physiological conditions of the living body at a specific position, an age, a height, a weight, a sex of the living body and other information inherent in the living body in addition to the physiological conditions of the living body. That is, it is very important that the cell or tissue for use in restoring a part of a living body conformed to the living body, and the culture environment can be set by use of information inherent to the living body serving as one element.
The method of cultivating a cell or tissue according to the seventh aspect of the invention is characterized in that the environment mimicking the living body under which the cell or tissue is cultivated is set by a nitrogen gas, an oxygen gas, a carbon dioxide gas respectively supplied through the culture medium, a temperature and a humidity. That is, since it is desirable that an environment under which the cell or tissue is cultivated corresponds to a living body, for example, if nitrogen gas, oxygen gas or carbon dioxide gas is supplied to a cultivation space and a temperature or humidity is set to that adapted for cultivation, a living body environment can be controlled to a desired state.
The method of cultivating a cell or tissue according to the eighth aspect of the invention is characterized in that the pressure applied to the cell or tissue can be arbitrarily set depending on the specific position of the living body. That is, it is possible to form an ideal or practical cell or tissue by applying a pressure, corresponding to the a living body at a specific position to be restored.
The method of cultivating a cell or tissue according to the ninth aspect of the invention is characterized in that the pressure applied to the cell or tissue is a pressure which is varied continuously, a pressure which is varied intermittently or a pressure which is varied periodically or a pressure combining these pressures. That is, it is possible to form the pressure pattern that is varied continuously, intermittently or periodically, and it is selected or combined to realize ideal physical stimulation so as to affect metabolism function, cell division cycle, concentration gradient or dispersion of living body stimulation so that the culture is enhanced.
The apparatus for cultivating a cell or tissue according to the tenth aspect of the invention is characterized in comprising a culture unit (culture circuit unit) having a culture chamber containing therein a cell or tissue and supplying culture medium, pressure application means (pressure application apparatus) for applying a pressure to the cell or tissue in the culture chamber, and culture medium supply means (culture medium supply apparatus) for intermittently or continuously supplying the culture medium to the culture unit.
That is, the culture unit accommodates the cell or tissue to be cultivated in the culture chamber to supply a culture medium needed for the cell or tissue that is intercepted from the open air. The cell or tissue that is intercepted from the open air is protected from contamination by bacteria and so forth, and hence it grows to a tissue having an excellent quality. A desired pressure by pressure application means in addition to physical stimulation caused by a hydraulic pressure and a flow by the culture medium is applied to the cell or tissue. As a result, it affects metabolism function, cell division cycle, concentration gradient or dispersion of living body stimulation so that the culture is enhanced. The mode of supply of the culture medium to the cell or tissue is arbitrarily set by the culture medium supply means, and the culture medium can be supplied to the cell or tissue intermittently or continuously so that the culture is enhanced by a variety of physical stimulation. The mode of the supply of the culture medium includes one of or both of the supply of a new culture medium at all times or the supply of the culture medium by repetitively circulating the culture medium. In the mode of circulation of the culture medium can save the culture medium, but there is an advantage of the prevention of the variation in concentration of the culture medium when supplying the culture medium in one direction.
The apparatus for cultivating a cell or tissue according to the eleventh aspect of the invention is characterized in further providing control means for controlling the pressure application means or culture medium supply means. That is, although the pressure application means or culture medium supply means can be controlled arbitrarily, various controls such as a feed back control or feed forward control and a program control and so forth can be performed by use of control means such as a computer. It is needless to say to add a personal collection control by an interruption, and the collection control is not excluded.
The apparatus for cultivating a cell or tissue according to the twelfth aspect of the invention is characterized in that the pressure applied from the pressure application means to the cell or tissue can be arbitrarily set depending on the cell or tissue. The manner of applying a pressure, namely, a pressure pattern is set, corresponding to a cell or tissue to be cultivated, thereby performing an efficient culture.
The apparatus for cultivating a cell or tissue according to the thirteenth aspect of the invention is characterized in that the pressure applied from the pressure application means to the cell or tissue is a pressure which is varied intermittently, a pressure which is repeated every given time or a pressure which increases or decreases every given time. That is, the pressure pattern can be conceived in all modes, thereby cultivating cell or tissue efficiently by selecting a mode of pressure pattern.
The apparatus for cultivating a cell or tissue according to the fourteenth aspect of the invention is characterized in that the culture unit is independent of and detached from a culture apparatus body. That is, the culture unit having the culture chamber for accommodating therein the cultivated cell or tissue can be independent of and detached from a culture apparatus body so that the cell or tissue can be moved together with the culture unit that is separated from the open air to protect the cell or tissue from being contaminated by bacteria during the motion thereof.
The apparatus for cultivating a cell or tissue according to the fifteenth aspect of the invention is characterized in that the culture unit is accommodated in a hermetically sealed space that is intercepted from an open air. That is, since the hermetically sealed space is the culture space, and it is intercepted from an open air, it is possible to set a culture environment by the supply of the desired gas, to protect the cell or tissue from the contamination by the open air.
The apparatus for cultivating a cell or tissue according to the sixteenth aspect of the invention is characterized in that the culture apparatus further comprises gas absorption means capable of absorbing a nitrogen gas, an oxygen gas, a carbon dioxide gas. That is, a nitrogen gas, an oxygen gas, a carbon dioxide gas can be supplied to the culture unit accommodated in the hermetically sealed space and the gas absorption means is provided in the culture unit so that the gas is applied to the cell or tissue and a living environment can be mimicked by supplying and controlling gas.
The apparatus for cultivating a cell or tissue according to the seventeenth aspect of the invention is characterized in that the hermetically sealed space is filled with a nitrogen gas, an oxygen gas, a carbon dioxide gas. That is, when a nitrogen gas, an oxygen gas, a carbon dioxide gas is filled in the culture space formed by the hermetically sealed space, a living body environment can be mimicked.
The apparatus for cultivating a cell or tissue according to the eighteenth aspect of the invention is characterized in further comprising a culture medium tank for storing therein the culture medium to be supplied to the culture unit. That is, the culture medium supply source is needed for supplying or circulating a necessary culture medium to the culture unit, and the culture medium tank is a supply source. Particularly, it is possible to prevent the culture medium held in the culture unit from being contaminated, if the culture medium tank is installed in the hermetically sealed space that is intercepted from the open air.
The apparatus for cultivating a cell or tissue according to the nineteenth aspect of the invention is characterized in that the culture chamber includes a pressure transmitting film for receiving a pressure from the outside. That is, it is possible to apply pressure application stimulation to the cell or tissue accommodated in the culture chamber in a state wherein it is intercepted from an open air, and to realize desired pressure application stimulation such as stimulation mimicking a living body environment by providing the pressure transmitting film.
The apparatus for cultivating a cell or tissue according to the twentieth aspect of the invention is characterized in that the culture chamber includes pressure buffering means. That is, it is possible to realize physical stimulation close to a living body environment and to enhance the culture of the cell or tissue by regulating a pressure by pressure buffering means when a part of a culture unit is pressurized.
The apparatus for cultivating a cell or tissue according to the twenty-first aspect of the invention is characterized in that the apparatus in the tenth aspect of the invention further comprises a pressure chamber fixed to the culture chamber by way of a pressure transmitting film, and a pressure is applied to the cell or tissue in the culture chamber by allowing a hydraulic pressure, an oil pressure or an air pressure to act on the cell or tissue in the culture chamber. That is, it is possible to realize desired pressure application stimulation and to mimic a living body environment with high accuracy by using any of the hydraulic pressure, the oil pressure or the air pressure as pressure forming means.
The apparatus for cultivating a cell or tissue according to the twenty-second aspect of the invention is characterized in that the culture medium supply means comprises a medium supply chamber provided in the culture unit and a medium supply unit for pressuring a culture medium that is taken in the medium supply chamber and supplying the pressurized culture medium. That is, the culture medium supply means is means for supplying and circulating the culture medium in the culture unit, and it is formed of various types, for example, if it is formed of the medium chamber and the medium supply unit for pressuring a culture medium that is taken in the medium supply chamber, the amount of applied pressure can be controlled to set a desired amount of supply medium.
The apparatus for cultivating a cell or tissue according to the twenty-third aspect of the invention is characterized in that a relief valve is provided in the culture, and when a pressure of the culture medium exceeds a given pressure which is arbitrarily set to the relief valve, the relief valve is opened to decrease the pressure of the culture medium. That is, it is important to buffer the pressure to be applied to the culture for applying ideal pressure application stimulation to the cell or tissue. If the pressure relieve valve is used as one means, and it is opened to decrease the pressure of the culture medium when the pressure of the culture medium exceeds a given pressure which is arbitrarily set to the relief valve, the culture medium is controlled in an ideal pressure state without contaminating the culture medium.
The apparatus for cultivating a cell or tissue according to the twenty-fourth aspect of the invention is characterized in that heating means or humidifying means are provided in a hermetically sealed space and the hermetically sealed space is kept and controlled at a desired temperature or humidity. That is, it is possible to provide a culture space conforming to a living body environment by controlling a temperature and a humidity of the hermetically sealed space in which the culture unit is accommodated.
The apparatus for cultivating a cell or tissue according to the twenty-fifth aspect of the invention is characterized in that a sound producing unit for applying a super-sound wave or the like sound wave in the culture chamber in the culture unit. That is, it is possible to mimic a living body environment acoustically by using the sound producing unit together because a living body receives acoustic stimulation from the outside, and possible to inject the cell or tissue to be cultivated in a culture chamber by use of a super-sound wave together with high reliability.
The apparatus for cultivating a cell or tissue according to the twenty-sixth aspect of the invention is characterized in that the apparatus further comprises control means for controlling concentration of a gas to be supplied to the hermetically sealed space. That is, it is possible to mimic a living body environment to enhance the culture of the cell or tissue by controlling the concentration of a gas to be supplied to the hermetically sealed space by controlling means.
The objects, features and advantages of the invention are now made more clear with reference to the following first to fourth embodiments of the invention, detail descriptions thereof and the attached drawings.